Synthesis, StructUre and Replication of Carcirogen-Modified Oligonucleotides

致癌物修饰寡核苷酸的合成、结构和复制

• 批准号: 8369307
• 负责人: Carmelo J Rizzo
• 金额: $ 34.05万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-15 至 2014-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8369307
• 关键词: 2-aminofluorene; Ames Assay; Amino Acids; Aromatic Amines; Binding; Biochemical; Biological; Biological Assay; Biological Process; Bypass; Calculi; Carcinogens; Chemicals; Colorectal Cancer; Complement; Complex; CpG dinucleotide; DNA; DNA Adducts; DNA Binding; DNA Damage; DNA Modification Process; DNA Sequence; DNA-Directed DNA Polymerase; Diet; Dinucleotide Repeats; Disease; Domestic Fowls; Dose; Epidemiologic Studies; Etiology; Exposure to; Family; Fishes; Foundations; Frameshift Mutation; Health; Heterocyclic Amines; Human; In Vitro; Laboratory Animals; Lead; Length; Lesion; Link; Malignant Neoplasms; Malignant neoplasm of prostate; Meat; Minor; Modeling; Molecular; Molecular Conformation; Mutagenesis; Mutagens; National Toxicology Program; Nature; Nucleotide Excision Repair; Oligonucleotides; Play; Polymerase; Population; Positioning Attribute; Predisposition; Proteins; Recombinants; Repair Enzymology; Research; Resources; Risk; Rodent; Role; Site; Smoking; Structure; Techniques; Testing; Time; Tumor-Suppressor Gene Inactivation; Tumorigenicity; Work; Xeroderma Pigmentosum; adduct; analog; base; carcinogenesis; chemical carcinogenesis; chemical property; chemical synthesis; citrate carrier; cooking; design; detoxication; exposed human population; genotoxicity; human tissue; in vitro testing; in vivo; interdisciplinary approach; interest; malignant breast neoplasm; nonhuman primate; professor; programs; public health relevance; red meat consumption; repaired; structural biology; sugar; tumorigenic

DESCRIPTION (provided by applicant): The covalent modification of DNA is believed to be the initial step in chemical carcinogenesis. Exposure to carcinogens is often a result of environmental or work conditions, diet or smoking. In recent years, a number of heterocyclic amines (HCAs) have been isolated from cooked meats; the HCAs are highly mutagenic in bacterial assays and potent haptocarcinogens in rodents and non-human primates. HCA-DNA adducts have been detected from human tissue after exposure to dietary relevant doses. Human exposure to HCAs is widespread and usually occurs over a lifetime; epidemiological studies have linked red meat consumption with increased risk of colorectal, breast, and prostate cancers. As such, many HCAs have been classified as "reasonably anticipated to be human carcinogens" by the National Toxicology Program and are likely to play a significant role in diet related human cancers. We have proposed an extensive program that interactively uses chemical synthesis, structural biology, polymerase enzymology, and DNA repair studies to define the influence of local DNA sequence and the chemical nature of HCA-adducts on the etiology of HCA carcinogenesis. A foundation of this program is the ability of the PIs lab to synthesize oligonucleotides containing structurally defined C8- and N2-dG adducts of HCAs (Aim 1). Many HCAs are potent inducers of frameshift mutations and we will focus on HCA-adducts in frameshift prone sequences. Frameshift inactivation of tumor suppressor genes is observed in many human colorectal cancers. The conformation of duplex DNA containing HCA adducts will be studied by a variety of techniques including multi-dimensional NMR; the HCA-modified duplexes will be examined opposite dC in a full-length complement strand (Aim 2) as well as opposite a two-base deletion (Aim 3). In addition, we will pursue crystallographic analysis of pre- and post-insertion complexes of HCA-modified duplexes bound to the model Y-family polymerase Dpo4. A key hypothesis of this program is that the precise conformation of the slipped mutagenic intermediate will correlate to the adduct's propensity to induced frameshifts. This will be tested by in vitro replication studies of HCA-modified templates using prokaryotic and human Y-family DNA polymerases (Aim 4). The extension products will be sequenced using an LC-ESI-MS-MS analysis developed at Vanderbilt. Modified DNA templates and primers designed to mimic discrete intermediates in the frameshift mechanism will also be studied. Finally, we propose to correlate the conformation of the HCA adducts with lesion recognition by the repair proteins UvrA and XPC7HR23B (Aim 5).

描述（由申请人提供）：DNA的共价修饰被认为是化学致癌作用的初始步骤。接触致癌物质通常是环境或工作条件、饮食或吸烟的结果。近年来，一些杂环胺（HCAs）已被分离出熟肉制品，HCAs是高度致突变性的细菌试验和有效的haptocarcinogenes在啮齿动物和非人类灵长类动物。在暴露于饮食相关剂量后，从人体组织中检测到HCA-DNA加合物。人类接触HCAs是普遍的，通常发生在一生中;流行病学研究已经将红肉消费与结直肠癌，乳腺癌和前列腺癌的风险增加联系起来。因此，许多HCA已被国家毒理学计划归类为“合理预期的人类致癌物”，并可能在与饮食有关的人类癌症中发挥重要作用。我们已经提出了一个广泛的计划，交互使用化学合成，结构生物学，聚合酶酶学和DNA修复的研究，以确定当地的DNA序列和HCA加合物的化学性质对HCA致癌的病因学的影响。该计划的基础是PI实验室合成含有结构确定的HCA的C8-和N2-dG加合物的寡核苷酸的能力（目标1）。许多HCA是移码突变的有效诱导剂，我们将重点关注移码倾向序列中的HCA加合物。在许多人类结直肠癌中观察到肿瘤抑制基因的移码失活。将通过多种技术（包括多维核磁共振）研究含有HCA加合物的双链DNA的构象;将在全长互补链中相对于dC（目标2）以及相对于两个碱基缺失（目标3）检查HCA修饰的双链体。此外，我们将追求前和后插入复合物的HCA修饰的双链体结合到模型Y-家族聚合酶DPO 4的晶体学分析。该程序的一个关键假设是，滑移致突变中间体的精确构象将与加合物诱导移码的倾向相关。这将通过使用原核和人Y家族DNA聚合酶的HCA修饰的模板的体外复制研究来测试（Aim 4）。将使用范德比尔特开发的LC-ESI-MS-MS分析对延伸产物进行测序。还将研究设计用于模拟移码机制中离散中间体的修饰DNA模板和引物。最后，我们建议将HCA加合物的构象与修复蛋白UvrA和XPC 7 HR 23 B的损伤识别相关联（目的5）。